Method for ligating nucleic acids and molecular cloning

ABSTRACT

The invention provides methods of covalently joining nucleic acid molecules and methods of molecular cloning. The methods provide either sequential or simultaneous ligation of flanking or vector nucleic acid molecules to nucleic acid insert molecules by topoisomerase and DNA ligase. The methods provide for directional and non-directional covalent joining and cloning of nucleic acid molecules.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/057,050, filed Jan. 25, 2002, which will issue as U.S. Pat. No.7,109,178 on Sep. 19, 2006, and which is a Continuation of U.S.application Ser. No. 09/513,710, filed Feb. 25, 2000, the entireties ofeach of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to methods of covalently joining nucleic acidmolecules and methods of molecular cloning of nucleic acid molecules.

BACKGROUND OF THE INVENTION

Construction of recombinant nucleic acid molecules requires twoenzymatic steps. First, site-specific restriction endonuclease digestionor PCR amplification are used to generate linear nucleic acid moleculeswith defined termini. Second, the linear molecules are covalently joinedat their termini in the presence of a ligase enzyme. Methods ofcovalently joining and cloning nucleic acid molecules that require onlyone step or that eliminate the use of restriction endonucleases orligases would be advantageous over the traditional method ofconstructing recombinant nucleic acid molecules.

SUMMARY OF THE INVENTION

It is an object of the invention to provide methods of covalentlyjoining nucleic acid molecules. It is a further object of the inventionto provide methods of cloning nucleic acid molecules. This and otherobjects of the invention are provided by one or more of the embodimentsdescribed below.

One embodiment of the invention provides a method of covalently joininga nucleic acid insert molecule to first and second nucleic acid flankingmolecules to form a ligated molecule. The method comprises incubatingthe insert molecule and the flanking molecules under conditions whichpermit their covalent joining to form a ligated molecule wherein aninsert molecule is positioned between the first and the second flankingmolecule. Each end of the insert molecule comprises a 5′-hydroxyl group.One end only of each of the first and second flanking moleculescomprises a covalently bound topoisomerase polypeptide.

Another embodiment of the invention provides a method of covalentlyjoining a nucleic acid insert molecule to first and second nucleic acidflanking molecules to form a ligated molecule. The method comprisesincubating an insert molecule, wherein one end of the insert moleculecomprises a 5′-hydroxyl group and the other end comprises a 5′-phosphategroup, with the first flanking molecule, wherein one end only of thefirst flanking molecule comprises a covalently bound topoisomerasepolypeptide. The incubation is done under conditions which permit theircovalent joining to form a ligated nucleic acid wherein the insertmolecule is positioned adjacent to the first flanking molecule. Thisligated nucleic acid is incubated with phosphatase under conditionswhich permit removal of a 5′-phosphate group from the ligated nucleicacid. The ligated nucleic acid is incubated with the second flankingmolecule. One end only of the second flanking molecule comprises acovalently bound topoisomerase polypeptide. The incubation is done underconditions which permit covalent joining to form a ligated moleculewhere the insert molecule is positioned between the first and the secondflanking molecule.

In still another embodiment of the invention a method of covalentlyjoining a nucleic acid insert molecule to first and second nucleic acidflanking molecules to form a ligated molecule is provided. The methodcomprises incubating an insert molecule and flanking molecules underconditions which permit their covalent joining to form a ligatedmolecule wherein an insert molecule is positioned between the first andthe second flanking molecule. One end of the insert molecule comprises a5′-hydroxyl group and the other end comprises a 5′-phosphate group. Oneend only of the first flanking molecule comprises a covalently boundtopoisomerase polypeptide and one end of the second flanking moleculecomprises a ligase substrate site.

Any of the first and second nucleic acid flanking molecules can togethercomprise a pair of left and right vector arms. Further, the ends of thevector arms not covalently joined to the insert can be covalently ornon-covalently joined to each other by a method selected from the groupconsisting of nucleic acid ligase mediated ligation, complementarysequence annealing, topoisomerase mediated ligation, in vitrosite-specific recombination, in vivo site-specific recombination, and invivo homologous recombination.

In still another embodiment of the invention a method of molecularcloning is provided. The method comprises incubating a nucleic acidinsert molecule comprising a 5′-hydroxyl group at one end and a5′-phosphate at the other end, and a linear cloning vector. The linearcloning vector comprises a covalently bound topoisomerase polypeptide atone end only and a ligation substrate site at the other end. Theincubation is done under conditions sufficient for their covalentjoining to form a ligated circular vector. The ligated circular vectoris transformed into a host cell.

Another embodiment of the invention provides a method for molecularcloning. The method comprises incubating a nucleic acid insert moleculewhere each end of the insert molecule comprises a 5′-hydroxyl group witha first and a second linear arm where one end only of each of the firstand second linear arms comprises a covalently bound topoisomerase andthe other end comprises a cloning substrate site. The incubation is doneunder conditions sufficient for their covalent joining to form a ligatedinsert molecule where the insert molecule is positioned between thefirst and the second linear arm. The ligated insert molecule istransformed into a host cell.

Even another embodiment of the invention provides a method for molecularcloning. A nucleic acid insert molecule, wherein one end of the insertmolecule comprises a 5′-hydroxyl group and the other end comprises a5′-phosphate group, and a first linear arm, wherein one end only of thefirst linear arm comprises a covalently bound topoisomerase polypeptideand the other end comprises a cloning substrate site are incubatedtogether. The incubation is done under conditions which permit theircovalent joining to form a ligated nucleic acid wherein the insertmolecule is positioned adjacent to the first linear arm. The ligatednucleic acid is incubated with phosphatase under conditions which permitremoval of a 5′-phosphate group from the ligated nucleic acid. Theligated nucleic acid is incubated with a second linear vector arm,wherein one end only of the second linear vector arm comprises acovalently bound topoisomerase polypeptide and the other end comprises acloning substrate site. The incubation is done under conditions whichpermit covalent joining to form a ligated insert molecule wherein theinsert molecule is positioned between the first and the second linearvector arm. The ligated insert molecule is transformed into a host cell.

In yet another embodiment of the invention a method for molecularcloning is provided comprising incubating a nucleic acid insertmolecule, wherein one end of the insert molecule comprises a 5′-hydroxylgroup and the other end comprises a 5′-phosphate group; a first lineararm, wherein one end only of the first linear arm comprises a covalentlybound topoisomerase polypeptide and the other end comprises a cloningsubstrate site; and a second linear arm, wherein one end of the secondlinear arm comprises a ligase substrate site and the other end comprisesa cloning substrate site. The incubation is done under conditions whichpermit their covalent joining to form a ligated insert molecule whereinthe insert molecule is positioned between the first and the secondlinear arm. The ligated insert molecule is transformed into a host cell.

The cloning substrate site can be selected from the group consisting ofa cos site, a LIC site, and a loxP site.

Where the cloning substrate site is loxP, the method can furthercomprise incubating in vitro the ligated insert molecule with a Crerecombinase and a circular plasmid comprising a loxP site. Theincubation is done under conditions sufficient for site-specificrecombination to form a circular plasmid comprising the ligated insertmolecule. The circular plasmid comprising the ligated insert molecule istransformed into a host cell.

Where the cloning substrate site is loxP the method can further comprisetransforming the ligated insert molecule into a host cell comprising acircular plasmid comprising a loxP site, wherein the cell expresses Crerecombinase. The transformation is done under conditions sufficient forsite-specific recombination to form a circular plasmid comprising theligated insert molecule within the cell.

Where the cloning substrate site is a site for homologous recombinationwith a circular plasmid vector the transformation step further comprisestransforming the ligated insert molecule into a host cell comprising acircular plasmid vector. The circular plasmid vector comprises a sitefor homologous recombination with the ligated insert molecule, and thehost cell is recA+. The transformation is done under conditionssufficient for homologous recombination to form a circular plasmidcomprising the ligated insert molecule within the host cell.

The first linear arm can comprise a left lambda arm comprising at oneend only a covalently bound topoisomerase. The second linear arm cancomprise a right lambda arm comprising at one end only a covalentlybound topoisomerase.

As used herein, the term “join” or “joining” refers to both covalent andnoncovalent attachment of one nucleic acid to another, or one end of anucleic acid to another end of a nucleic acid. “Covalent” joining refersto the attachment of one end of a nucleic acid strand to another end ofa nucleic acid strand via a phosphate bond or to attachment of one endof a double-stranded nucleic acid to another double-stranded end viaphosphate bonding on one or both strands. “Non-covalent” joining refersto attachment of one end of a nucleic acid to another end via annealingof a single-stranded regions to each other; that is, no phosphate bondis generated in this embodiment.

“Ligate” or “ligated” refers to the covalent joining of two ends of oneor more nucleic acid molecules.

“Complementary annealing” refers to annealing, or the pairing of bases,of complementary regions of one or more nucleic acids, and thus to theformation of hydrogen bonds and other non-covalent interactions betweenpairs of bases.

A “topoisomerase” is a polypeptide that is capable of covalently joiningto at least one strand of a nucleic acid molecule and ligating thatstrand to another strand, as described hereinbelow. Topoisomeraseaccording to the invention comprises type I topoisomerases.

“Bound to” refers to a covalent bonding of a topoisomerase polypeptideto a nucleic acid molecule.

“Nucleic acid molecule” refers to a double-stranded nucleic acid, unlessotherwise specified.

“One end only” refers to the presence of a topoisomerase polypeptide atone end of a nucleic acid molecule, where the nucleic acid moleculecontains two ends.

The term “site” is meant to designate a contiguous stretch ofnucleotides, e.g., 1-100 bases in length, usually 5-25 bases in length,e.g., 8-16 bases, that is susceptible to (i.e., a substrate for)modification by an enzyme that modifies nucleic acids, e.g., a ligase ora restriction enzyme.

A “cloning substrate site”, as used herein, is a site occurring on anucleic acid molecule for the covalent or non-covalent joining ofnucleic acid sequences or for recombination. Examples of cloningsubstrate sites include cos sites, LIC sites, sites for site-specificrecombination, such as lambda attachment elements or loxP sites, sitesfor homologous recombination, and ligation substrate sites.

A “ligation substrate site”, as used herein, is a site occurring on anucleic acid molecule of the invention that is capable of becomingcovalently joined to another nucleic acid molecule in the presence of aligase enzyme, such as DNA ligase.

A “vector arm” or a “linear arm”, as used herein, is a linear nucleicacid molecule, and is preferably a portion or fragment of abacteriophage or plasmid genome.

“Directional” cloning refers to a cloning method in which, by selectingsteps in the method, one can obtain a desired orientation of a givennucleic acid molecule upon cloning into another nucleic acid molecule orbetween two other nucleic acid molecules; as used herein, “orientation”may refer to 5′ to 3′ with reference to a given open reading frame or agiven control region or a known sequence. Thus, for example, an insertmolecule may contain an open reading frame having a 5′-3′ orientationwith respect to transcription and the insert molecule may bedirectionally cloned between a left and right vector arms such that theligated (cloned) molecule comprises, from 5′ to 3′: left vector arm, 5′insert 3′, right vector arm. “Non-directional” cloning refers to cloningmethods which produce a ligated molecule in which the insert, forexample, appears between the two arms in either orientation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the non-directional covalent joining of an insert moleculewith 5′-OH groups on each end to a right vector arm and a left vectorarm each comprising a topoisomerase polypeptide on one end only.

FIG. 2 shows the directional covalent joining of an insert molecule witha 5′-OH group on one end and a 5′-phosphate group on the other end to aright vector arm and a left vector arm each comprising a topoisomerasepolypeptide on one end only.

FIG. 3 shows the directional covalent joining of an insert molecule witha 5′-OH group on one end and a 5′-phosphate group on the other end to aleft vector arm comprising a topoisomerase polypeptide on one end onlyand a right vector arm comprising a ligation substrate site on one end.

FIG. 4 shows the directional cloning of an insert molecule with a 5′-OHgroup on one end and a 5′-phosphate group on the other end to a linearvector molecule. The linear vector molecule comprises a topoisomerasemolecule on one end only and a ligation substrate site on the other end.

FIG. 5 shows the non-directional cloning of an insert molecule with5′-OH groups on each end to a right vector arm and a left vector armeach comprising a topoisomerase polypeptide on one end only and acloning substrate site, cos, on the other end.

FIG. 6 shows the directional cloning of an insert molecule with a 5′-OHgroup on one end and a 5′-phosphate group on the other end to vectormolecules comprising a topoisomerase polypeptide on one end only and acloning substrate site, cos, on the other end.

FIG. 7 shows the non-directional cloning of an insert molecule with5′-OH groups on each end to a right vector arm and a left vector armeach comprising a topoisomerase polypeptide on one end only and acloning substrate site, LIC, on the other end.

FIG. 8 shows the directional cloning of an insert molecule with a 5′-OHgroup on one end and a 5′-phosphate group on the other end to vectormolecules comprising a topoisomerase polypeptide on one end only and acloning substrate site, LIC, on the other end.

FIG. 9 shows the directional cloning of an insert molecule with a 5′-OHgroup on one end and a 5′-phosphate group on the other end to vectormolecules comprising a topoisomerase polypeptide on one end only of alambda vector arm.

FIG. 10 shows the non-directional cloning of an insert molecule with5′-OH groups on each end to a right plasmid arm and a left plasmid armeach comprising a topoisomerase polypeptide on one end only.

FIG. 11 shows the directional cloning of an insert molecule with a 5′-OHgroup on one end and a 5′-phosphate group on the other end to vectormolecules comprising a topoisomerase polypeptide on one end only.

FIG. 12 shows the non-directional cloning of an insert molecule with5′-OH groups on each end to a right vector arm and a left vector armeach comprising a topoisomerase polypeptide on one end only and acloning substrate site, a loxP site, on the other end.

FIG. 13 shows the directional cloning of an insert molecule with a 5′-OHgroup on one end and a 5′-phosphate group on the other end to vectormolecules comprising a topoisomerase polypeptide on one end only and acloning substrate site, a loxP site, on the other end.

FIG. 14 shows the non-directional cloning of an insert molecule with5′-OH groups on each end to a right vector arm and a left vector armeach comprising a topoisomerase polypeptide on one end only and acloning substrate site, a site for homologous recombination, on theother end.

FIG. 15 shows the directional cloning of an insert molecule with a 5′-OHgroup on one end and a 5′-phosphate group on the other end to vectormolecules comprising a topoisomerase polypeptide on one end only and acloning substrate site, a site for homologous recombination, on theother end.

DETAILED DESCRIPTION OF THE INVENTION

Insert Polynucleotide Molecules

Insert polynucleotide molecules comprise isolated and purifieddouble-stranded DNA, double-stranded RNA, or double-stranded DNA/RNAhybrid nucleic acids. An insert molecule can be a full-length moleculeor a fragment of a full-length molecule. Further, an insert molecule canbe naturally-occurring, i.e., found in nature or recombinant.

Preferably, insert polynucleotides are isolated free of othercomponents, such as proteins and lipids. Insert polynucleotides can bemade by a cell and isolated or can be synthesized in the laboratory, forexample, using an automatic synthesizer or an amplification method suchas PCR. Where an insert polynucleotide is prepared by PCR, the insert isgenerated using a pair of primers comprising a 3′-primer and a5′-primer. Both the 3′-primer and the 5′-primer can comprise a5′-hydroxyl group to produce an insert with 5′-hydroxyl groups (5′-OH)on both ends. Alternatively, one of the primers can comprise a5′-hydroxyl group and one can comprise a 5′-phosphate group to producean insert with a 5′-OH group on one end and a 5′-phosphate (5′-P) groupon the other end. Optionally, both the 3′-primer and the 5′-primer cancomprise a 5′-phosphate group to produce an insert with 5′-P groups onboth ends.

Molecules Flanking an Insert Molecule

An insert polynucleotide molecule can be covalently joined to severaltypes of molecules, such as a double-stranded DNA, a double-strandedRNA, and a double-stranded DNA/RNA hybrid molecule. Preferably, aninsert polynucleotide molecule is covalently joined to a vector moleculeor to vector molecules such as a linear arm of a plasmid orbacteriophage. Vectors suitable for ligation of an insert moleculeinclude bacteriophage, such as bacteriophage lambda, including, but notlimited to lambda insertion vectors such as Lambda ZAP®II vector, ZAPExpress® vector, Lambda ZAP®-CMV vector (Stratagene), lambda gt10, andlambda gt11. Lambda replacement vectors, for example Lambda FIX®IIvector, Lambda DASH®II vector, and Lambda EMBL3 and Lambda EMBL4(Stratagene) can also be used as vectors.

Both prokaryotic and eukaryotic linear plasmids can be used as vectors.See e.g., Meinhardt et al. (1997) Appl. Microbiol. Biotechnol.47:329-36; Fukuhara, (1995) FEMS Microbiol. Lett. 131:1-9; Hinnebusch &Tilly, (1993) Mol Microbiol. 10:917-22. For example, the plasmidprophage N15 of E. coli is a suitable linear plasmid vector. See Rybchin& Svarchevsky (1999) Mol. Microbiol. 33:895-903.

Vector nucleic acid polynucleotides, such as bacteriophage and plasmidscan be isolated and purified from cells carrying these elementsaccording to methods well known in the art. See e.g. MOLECULAR CLONING:A LABORATORY MANUAL (Sambrook et al., eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1989) and Ausubel (CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., eds., John Wiley & Sons,New York, 1987)). Additionally, many bacteriophage and plasmid vectorsare commercially available. The bacteriophage or plasmid nucleic acidcan be prepared, if necessary by cleavage with an appropriaterestriction enzyme such that the digested bacteriophage or plasmidnucleic acid is compatible with an insert molecule.

Preferably, an insert molecule is covalently joined to right and leftlambda linear vector arms such that the insert molecule is positionedbetween right and left lambda linear vector arms. In lambda insertionvectors, a left vector arm can comprise lambda nucleic acids occurringto the left of the insertion site and a right vector arm can compriseslambda nucleic acids occurring to the right of the insertion. In lambdareplacement vectors, a left lambda arm comprises lambda nucleic acidoccurring to the left of the nucleic acids to be replaced by the insertnucleic acids and a right lambda arm comprises lambda nucleic acidsoccurring to the right of the nucleic acids to replaced by the insertnucleic acids. Lambda vectors can vary in nucleic acid sequence;however, the left arm can typically comprise the head and tail genesA-J, while the right arm can typically comprise from p_(R) through acosR site of a lambda genome.

Preferably, the vector or flanking molecule to which the insert is to becovalently joined is a linear molecule comprising a topoisomerasecovalently linked to only one end of the linear molecule. Adouble-stranded DNA, double-stranded RNA, or double-stranded DNA/RNAmolecule with one topoisomerase molecule bound to one end of the DNA orRNA molecule is a univalent molecule. DNA topoisomerases catalyze aconversion in the linking number of a double-stranded DNA molecule. Thelinking number is the number of times one DNA strand crosses over thesecond DNA strand in space. Type 1 topoisomerases act by making atransient break in one strand of a nucleic acid. A type 1 topoisomerasefirst binds to a nucleic acid and nicks one strand of the nucleic acid.A stable complex is formed where the free 3′-phosphate end of the nickedstrand is covalently bound to a tyrosine residue of the enzyme. Thesecond strand is pulled through the gap in the first strand and the gapis then sealed by the enzyme. The gap can be sealed at the same bondoriginally nicked or the complex can combine with a heterologous nucleicacid, such as an insert molecule, that has a 5′-hydroxy end. Where thecomplex is combined with a heterologous nucleic acid, a recombinantmolecule is formed.

Type 1 topoisomerases include, but are not limited to E. colitopoisomerase I (Keck et al., (1999) Nat. Strut. Biol. 6:900), E. colitopoisomerase III (Mondragon et al., (1999) Structure Fold. Des.7:1373), S. cerevisiae topoisomerase III (Kim et al., (1992) J. Biol.Chem. 267:17178), human topoisomerase III (Hanai et al., (1996) Proc.Natl. Acad. Sci. 93:3653), the type I topoisomerase from chloroplasts(Mukherjee et al. (1994) 269:3793; Fukata et al. (1991) J. Biochem(Tokyo) 109:127), thermophilic reverse gyrases (Nadal et al., (1994) J.Biol. Chem. 269:5255; Slesarev et al., (1991) J. Biol. Chem. 266:12321;Bouthier de la Tour et al., (1991) J. Bact. 173:3921), thermophilic D.amylolyticus topoisomerase III (Slesarev et al., (1991) J. Biol. Chem.266:12321), and vaccinia DNA topoisomerase I (Shuman et al., (1987)Proc. Natl. Acad. Sci. 84:7478). Site-specific type I DNA topoisomerasesare particularly useful in the invention. Site-specific type I DNAtopoisomerases include vaccinia topoisomerase and pox virustopoisomerases.

A topoisomerase enzyme can be covalently linked to a vector or flankingmolecule by, for example, the method of Heyman et al. (Genome Res.(1999) 9:383). Briefly, Vaccinia DNA topoisomerase cleaves thephosphodiester backbone of one strand of a nucleic acid at a consensuspentopyrimidine element: 5′-C/TCCTT-3′ (SEQ ID NO:1). This element canbe added onto the end of a vector or flanking molecule. Vacciniatopoisomerase can then be incubated with the vector or flanking moleculesuch that the topoisomerase becomes covalently bound to the underlined Tin the C/TCCTT sequence. Optionally, nuclease treatment, such asexonuclease III treatment can be used to remove single strand ends fromthe element such that a blunt-ended insert fragment with topoisomerasebound to the molecule is formed.

Optionally, the molecule to which the insert is to be covalently joinedis a linear molecule comprising a ligation substrate site at a first endof the linear molecule. A ligation substrate site comprises a site fornucleic acid ligation that is mediated by a ligase enzyme. A ligationsubstrate site can comprise any double-stranded nucleic acid that hasblunt ends or protruding termini that can be covalently joined toanother nucleic acid molecule in the presence of a ligase enzyme.Preferably, the ligation substrate site comprises a 5′-phosphate groupand is complementary to one end of an insert molecule. A ligationsubstrate site can be produced by, for example cleaving adouble-stranded nucleic acid molecule with a restriction enzyme thatproduces blunt-ended termini, 5′-protruding ends, or 3′-protruding endsand purifying the nucleic acid molecule. A ligation between a linearmolecule comprising a ligation substrate site and an insert moleculetakes place in the presence of a ligase enzyme such as bacteriophage T4DNA ligase or Pfu DNA ligase (Stratagene). Preferably, the vector orflanking molecule to which the insert is to be covalently joined is alinear molecule comprising a topoisomerase covalently linked to only oneend of the molecule or a ligation substrate site at one end of thelinear molecule. The second end of the linear molecule preferablycomprises a cloning substrate site such as, a cos site, a LIC site, asite-specific recombination site (such as a loxP site or lambdaattachment element), a homologous recombination site or a ligationsubstrate site.

A bacteriophage lambda genome has cos sites at the ends of the genome.See, LAMBDA II (Roger W. Hendrix, ed., Cold Spring Harbor LaboratoryPress) 1983; Higgins et al., (1995) J. Mol. Biol. 252:31; Higgins etal., (1994) EMBO J. 13:6152; Cue et al., (1993) J. Mol. Biol. 234:594;Cue et al., (1993) Proc. Natl. Acad. Sci. USA 90:9290. Cleavage occursat a left cos site (as defined on a standard lambda map) to generate afree end that is inserted into a capsid. The insertion of nucleic acidcontinues until a right cos site is encountered. Cleavage occurs at theright cos site to generate the second end. Any nucleic acid moleculethat is contained between two cos sites can be packaged. A nucleic acidmolecule comprising a cos site, a fragment of a cos site, a mutant of acos site, or a variant of a cos site can be isolated from a preparationof bacteriophage lambda DNA or may synthesized in the laboratory. Anucleic molecule comprising a cos site can be ligated to the end of themolecule to which the insert is to be covalently joined. Alternatively,a cos site can be added to the end of a molecule to which the insert isto be covalently joined using standard molecular biology cloningtechniques such as PCR. In the methods of the invention distal ends(i.e., the ends of vector arms not covalently joined to an insertmolecule) of vector arms containing terminal cos sites can be readilyannealed to one another in E. coli host cells by virtue of theirexplicit sequence. cos sites do not appreciably anneal in vitro at roomtemperature.

A ligation-independent cloning (LIC) site can be any size, but ispreferably 12 to 13 nucleotides or longer. Sites longer than 12-13nucleotides may work more efficiently, e.g., up to 24 bases, or up to 48bases or longer. See Aslanidis and de Jong, (1990) Nucleic Acids Res.18:6069. The 12-13 nucleotide terminus can comprise any nucleic acidsequence; however, preferably one or none of the nucleotides of a 3′strand of the 12-13 nucleotide terminus is an adenosine. A nucleicmolecule comprising a LIC site can be ligated to the ends of the vectoror flanking molecule to which the insert is to be covalently joined.Alternatively, a LIC site can be added to the end of a vector orflanking molecule to which the insert is to be covalently joined usingstandard molecular biology cloning techniques, such as by PCR.

Where the second end of a linear molecule comprises a LIC site, aligated insert/vector molecule will be formed that comprises LIC ends ateach end of the ligated insert/vector molecule. The insert can then bejoined to a LIC ready vector. Aslanidis et al., (1994) PCR Methods Appl.4:172; Aslanidis and de Jong (1990) Nucleic Acids Res. 18:6069. Briefly,the ligated insert/vector molecule is subjected to treatment with, forexample, Pfu DNA polymerase in the presence of dATP. In the absence ofdTTP, dGTP, and dCTP, the 3′- to 5′-exonuclease activity of Pfu DNApolymerase removes 12 to 13 nucleic acids from the 3′-ends of theligated insert/vector molecule. This activity continues until the firstadenine is encountered. This produces a ligated insert/vector moleculewith 5′-extended single-stranded tails that are complementary to thesingle-stranded tails of a LIC ready vector. The ligated insert/vectormolecule will anneal to the LIC ready vector without further enzymatictreatment.

The second end of the linear molecule can further comprise a site forhomologous recombination or a site for site-specific recombination.Homologous recombination is a recombination event occurring betweenhomologous sequences of nucleic acids. The enzymes responsible forhomologous recombination can use any pair of homologous sequences assubstrates, although some types of nucleic acid sequences can be favoredover others. Sites for homologous recombination comprise nucleic acidsequences that are homologous to the nucleic acid sequences of a cloningvector, such as a circular plasmid. The sites can insert (or integrate)into a cloning vector by homologous recombination, thereby inserting ordisplacing a nucleic acid sequence, or deleting a nucleic acid sequencealtogether.

To create a homologous recombinant plasmid cloning vector, a plasmidcloning vector is prepared which contains homologous recombinationnucleic acid sites that are substantially homologous to those sitesoccurring on the ligated insert/vector of interest. Substantiallyhomologous nucleic acid sequences are those nucleic acid sequences thatshare sufficient nucleic acid sequence homology to provide forsufficient homologous recombination between a ligated insert/vectorsequence and a plasmid cloning vector. Sufficient nucleic acid sequencehomology is the amount which provides for homologous recombination at afrequency which allows for detection of plasmid cloning vectors in whichhomologous recombination and integration of the ligated vector/inserthas occurred. Substantially homologous nucleic acid sequences preferablyshare regions with about 60% to 100% nucleic acid sequence homology, andmore preferably about 75% to 100% homology in the nucleic acid sequence.A site for homologous recombination can be present in the plasmidcloning vector in two or more copies. The homologous recombination sitesin the plasmid cloning vector are of sufficient length for successfulhomologous recombination with a ligated insert/vector molecule.Typically, each homologous recombination site is at least 30, 75, 100,150, 250, 500, or 1000 base pairs. The ligated insert/vector sequencecomprises these substantially homologous recombination sites at both the5′- and 3-′ ends. The ligated insert/vector sequence is transformed intoa host cell, such as an E. coli cell that contains the plasmid cloningvector. Preferably, the host cell is RecA+. Rec A is the product of therecA locus of E. coli and is a protein that is involved inrecombination.

In addition to homologous recombination as described above,enzyme-assisted site-specific integration systems are known in the artand can be applied to integrate a ligated nucleic acid insert/vectormolecule at a predetermined location in a cloning vector molecule.Site-specific recombination is a recombination event between specificpairs or sequences. The recombination event involves specific sequencesof nucleic acids comprising a short stretch of homology necessary forthe recombination event. The enzymes involved in the recombination eventwill act only on this particular pair of target sequences. Examples ofsuch enzyme-assisted integration systems include the Crerecombinase/loxP target system (e.g., as described in Baubonis and Sauer(1993) Nucl. Acids Res. 21:2025; and Fukushige and Sauer, (1992) Proc.Natl. Acad. Sci. USA 89:7905). A loxP site (locus of crossing over)comprises two 13 base pair inverted repeats separated by an 8 base pairasymmetric spacer region: ATAACTTCGTATA ATGTATGC TATACGAAGTTAT (SEQ IDNO:2)

A loxP site of the invention comprises variants and mutants of thissequence that function to produce site-specific recombination. Cre is a38 kDa recombinase protein from bacteriophage P1 which mediatesintramolecular and intermolecular site-specific recombination betweenloxP sites. Sauer, (1993) Methods Enzymol. 225:890. A loxP site is anasymmetrical nucleotide sequence and two lox sites on the same DNAmolecule can have the same or opposite orientation with respect to oneanother. See U.S. Pat. No. 4,959,317. Where two loxP sites occur in thesame orientation on a nucleic acid molecule, recombination between theloxP sites results in the deletion of the nucleic acid segment locatedbetween the two loxP sites and a connection between the resulting endsof the original nucleic acid molecule. The deleted nucleic acid moleculewill form a circular molecule of nucleic acid. The original nucleic acidmolecule and the circular nucleic acid molecule will each contain asingle loxP site. Where two loxP sites occur in opposite orientations onthe same nucleic acid molecule recombination will result in an inversionof the nucleotide sequence of the nucleic acid segment located betweenthe two loxP sites. Further, where two loxP sites occur on each of twonucleic acid segments, reciprocal exchange of nucleic acid segmentsproximate to the loxP sites can occur.

Methods of Covalently Joining

Insert polynucleotide molecules comprising a 5′-OH group on each end ora 5′-OH on one end and a 5′-phosphate group on the other end can becovalently joined to flanking polynucleotide molecules such thatnon-directional or directional covalent joining is achieved. Where aninsert polynucleotide molecule has a 5′-OH group on each endnon-directional covalent joining of the insert to flankingpolynucleotide molecules results. For example, where an insertpolynucleotide (I) with a 5′-OH group at each end is covalently joinedto flanking molecules, for example, a left vector arm (LVA) and a rightvector arm (RVA) each with a topoisomerase polypeptide covalently joinedat only one end, the result will be non-directional covalent joining ofthe molecules. A LVA, RVA, and insert molecule are incubated togetherunder conditions sufficient to permit their topoisomerase-mediatedcovalent joining to form a covalently joined nucleic acid molecule wherethe insert molecule is positioned between the LVA and RVA. Fourdifferent covalently joined products (ligated insert/vector molecules)will result: LVA-I-LVA, RVA-I-RVA, LVA-I-RVA, and RVA-I-LVA. Only theLVA-I-RVA and RVA-I-LVA products are viable replication competententities. Where an insert polynucleotide has a 5′-OH group on one endand a 5′-phosphate group on the other end directional covalent joiningof the insert to flanking polynucleotide molecules can result. Forexample, where an insert polynucleotide is covalently joined to aflanking molecules such as a LVA and a RVA, each comprising atopoisomerase covalently bound to only one end, directional covalentjoining of the molecules can result. A first vector arm, for example, aLVA is covalently joined to an insert molecule at the 5′-OH end byincubating a LVA and an insert molecule together under conditionssufficient to permit topoisomerase-mediated covalent joining to form aligated nucleic acid molecule where the insert molecule is positionedadjacent to a LVA to create LVA-I-phosphate. The 5′-phosphate end of theinsert is unable to be ligated to the LVA or RVA because either the LVAor RVA has a 3′-phosphate, which is the site to which a topoisomerasepolypeptide is joined to the LVA and RVA. The LVA-I-5′-phosphate istreated with phosphatase, under conditions which permit removal of a5′-phosphate group from the ligated nucleic acid resulting in aLVA-I-5′-OH molecule. The LVA-I-5′-OH molecule is then covalently joinedto the RVA to form LVA-I-RVA by incubating a LVA-I-5′-OH molecule with aRVA under conditions which permit topoisomerase covalent joining to forma ligated molecule where the insert molecule is positioned between a RVAand a LVA (a ligated insert/vector molecule).

Alternatively, an insert polynucleotide comprising a 5′-OH group on oneend and a 5′-phosphate group on the other end can be covalently joinedin a directional manner to a flanking nucleic acid molecule comprising atopoisomerase polynucleotide on only one end and to a second flankingmolecule comprising a ligation substrate site on one end. For example,an insert molecule can be covalently joined to a flanking nucleic acidmolecule, such as a LVA, comprising a topoisomerase polypeptide on onlyone end and to, for example, a RVA comprising a ligation substrate endon one end. The insert LVA, and RVA are covalently joined bytopoisomerase-mediated joining and ligase-mediated joining underconditions sufficient to form a ligated nucleic acid where the insertmolecule is positioned between a LVA and a RVA to form LVA-I-RVA (aligated insert/vector molecule). This reaction can take place in onestep.

After the ligated insert/vector molecule described above has beenconstructed, the two vector arms can be non-covalently or covalentlyjoined to one another, at the ends distal to the covalently attachedtopoisomerase polypeptide or ligation substrate site (i.e., at theirfree ends), by a number of methods such that a circular molecule isformed. For example, the ends of the ligated insert/vector molecule cancomprise ligase substrate sites or complementary nucleic acid sequencessuch that the ends can be joined by ligase enzyme mediated ligation orcomplementary sequence annealing. Further, where the ends of the ligatedinsert/vector molecule comprise 5′-OH groups the ends can be joined bytopoisomerase mediated ligation using a polynucleotide comprising atopoisomerase polypeptide at both ends of the polynucleotides. See e.g.U.S. Pat. No. 5,766,891. Further, where the ends of the ligatedinsert/vector molecule comprise in vitro or in vivo site-specificrecombination sites or in vivo homologous recombination sites theligated insert/vector molecule can be recombined into a circular plasmidcontaining the same recombination sites.

The methods of directional and non-directional covalently joining ofnucleic acid molecules are useful in, for example, end-labeling, ligandtagging, and molecular cloning.

Methods of Molecular Cloning

Insert polynucleotide molecules comprising a 5′-OH group on each end ora 5′-OH on one end and a 5′-phosphate group on the other end can becloned into vector molecules such that non-directional or directionalcloning is achieved.

Non-directional cloning can be accomplished by cloning a polynucleotideinsert molecule comprising 5′-OH groups at both ends of the moleculeinto a nucleic acid vector. For example, an insert polynucleotide (I)with a 5′OH group at each end can be cloned into a vector, such as aleft vector arm (LVA) and a right vector arm (RVA) where each vector armhas a topoisomerase polypeptide covalently joined at only one end of thevector arm. The result will be non-directional covalent joining of themolecules. Preferably, the LVA and RVA molecules have a cloningsubstrate site, such as a cos site, a LIC site, a loxP site, a site forhomologous recombination, a site for site-specific recombination, or aligase substrate site at the other end of the molecule. A LVA, RVA, andinsert molecule are incubated together under conditions sufficient fortopoisomerase-mediated covalent joining of the molecules to form aligated nucleic acid wherein the insert molecule is positioned betweenthe LVA and RVA. Four different covalently joined products will result:LVA-I-LVA, RVA-I-RVA, LVA-I-RVA, and RVA-I-LVA (ligated insert/vectormolecules). Only the LVA-I-RVA and RVA-I-LVA products are viablereplication competent entities.

Directional cloning can be accomplished by cloning a polynucleotideinsert molecule comprising a 5′-OH group at one end of the molecule anda 5′-phosphate group at the other end into a nucleic acid vector. Forexample, an insert polynucleotide (I) with a 5′-OH group at one end anda 5′-phosphate at the other end can be cloned into a linear cloningvector, where the linear cloning vector has a topoisomerase polypeptidecovalently joined at one end and a ligation substrate site at the otherend. The insert polynucleotide, the linear cloning vector, and a ligaseare incubated together under conditions sufficient for their covalentjoining to form a ligated circular vector (a ligated insert/vectormolecule). The circular vector can then be transformed into a host cell.

Directional cloning can also be accomplished by cloning an insertpolynucleotide having a 5′-OH group on one end and a 5′-phosphate groupon the other end into a vector where the vector comprises, for example,two vector arm molecules comprising a topoisomerase polynucleotide atonly one end and a cloning substrate site at the other end. For example,a first vector arm, LVA, is covalently joined to an insert molecule atthe 5′-OH end by incubating a LVA and an insert molecule together underconditions sufficient to permit topoisomerase-mediated covalent joiningto form a ligated nucleic acid molecule where the insert molecule ispositioned adjacent to a LVA to create LVA-I-phosphate. The 5′-phosphateend of the insert is unable to be ligated to the LVA or RVA because atopoisomerase polypeptide is joined to the LVA and RVA at the5′-phosphate. The LVA-I-5′-phosphate is treated with phosphatase, underconditions which permit removal of a 5′-phosphate group from the ligatednucleic acid resulting in a LVA-I-5′-OH molecule. The LVA-I-5′-OHmolecule is then covalently joined to the RVA to form LVA-I-RVA byincubating a LVA-I-5′-OH molecule with a RVA under conditions whichpermit topoisomerase covalent joining to form a ligated molecule wherethe insert molecule is positioned between a RVA and a LVA (a ligatedinsert/vector molecule). Preferably, the cloning substrate site is a cossite, a LIC site, a loxP site, a site for homologous recombination, asite for site-specific recombination, or a ligation substrate site.

Alternatively, directional cloning can be accomplished with an insertpolynucleotide comprising a 5′-OH group on one end and a 5′-phosphategroup on the other end and two vector molecules. One vector moleculecomprises a topoisomerase polynucleotide on only one end and a cloningsubstrate site on the other end. The other vector molecule comprises aligation substrate site on one end and a cloning substrate site on theother end. An insert, a first vector molecule comprising a topoisomerasepolypeptide at one end and a cloning substrate site at the other end,such as a LVA, and a second vector molecule such as a RVA comprising aligation substrate site at one end and a cloning substrate at the otherend are covalently joined by topoisomerase-mediated joining andligase-mediated joining under conditions sufficient to form a ligatednucleic acid where the insert molecule is positioned between the LVA andthe RVA vector molecules (a ligated insert/vector molecule). Preferably,the cloning substrate site is a cos site, a LIC site, a loxP site, asite for homologous recombination, a site for site-specificrecombination, or a ligation substrate site.

Where the ligated insert/vector molecule comprises cos sites at eachend, the linear molecule can be transformed directly into a host cell.Where the ligated insert/vector molecule comprises LIC ends at each end,the LIC ends can be annealed to a circular plasmid vector with LICcompatible ends. The circular molecule can be transformed into a hostcell. Where the ligated insert/vector molecule comprises loxP sites onboth ends, the ligated insert/vector molecule can be recombined into acircular plasmid in vitro in the presence of Cre recombinase. Therecombinant circular plasmid can then be transformed into a host cell.Alternatively, a ligated insert/vector molecule with loxP sites at bothends of the molecule can be directly transformed into a host cell, suchas E. coli harboring a plasmid suitable for site-specific recombination.The host cell may be rec A+ or recA−, and is preferably recA−. Where thecovalently joined insert/vector molecule comprises sites for homologousrecombination at each end, the covalently joined insert/vector moleculecan be directly transformed into a suitable host cell harboring aplasmid suitable for homologous recombination.

The covalently joined insert/vector can be transformed into aprokaryotic or eukaryotic cell. Preferably, the covalently joinedinsert/vector is transformed into a prokaryotic host cell, such as abacteria cell such as E. coli. Transformation of a ligated insert/vectormolecule into a host cell can be done by any method known in the art.Methods for transformation of host cells can be found in Sambrook et al.and Ausubel and include, but are not limited to transfection, chemicaltransformation, electroporation, and lipofection. Where a bacteriophagelambda vector has been used according to the invention, the ligatedinsert/lambda vector can be packaged in vitro and then transfected intohost cells, such as XL1-Blue E. coli. See e.g. Sambrook et al.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

EXAMPLES Example 1

Inter-Molecular Ligation and Molecular Cloning Using UnivalentTopoisomerase-Bound DNA

An insert nucleic acid molecule, for example, a PCR product, can begenerated by PCR using a primer set consisting of a 5′-primer and3′-primer. Two vector nucleic acid molecules, for example, a left vectorarm and a right vector arm are prepared such that a topoisomerase enzyme(TOPO) is covalently bound only to one end of a nucleic acid molecule toform a univalent topoisomerase vector molecule. PCR primers forgenerating an insert molecule can be synthesized to possess either ahydroxyl group or phosphate group at each of the 5′-ends. A hydroxylgroup permits ligation to topoisomerase-bound DNA while a phosphategroup prohibits such ligation.

For non-directional ligation of a PCR insert molecule to, for exampletwo vector arms, both PCR primers will possess 5′-hydroxyl groups. ThePCR insert can ligate with the vector arms to form four different typesof ligation products: 1) left vector arm (LVA)-insert molecule (I)-leftvector arm (LVA); 2) right vector arm (RVA)-insert (I)-right vector arm(RVA); 3) LVA-I-RVA; and 4) RVA-I-LVA. Only the LVA-I-RVA and RVA-I-LVAcreate viable replication competent entities (FIG. 1).

For directional ligation of a PCR insert molecule to, for example, leftand right vector arms, one PCR primer possesses a 5′-hydroxyl group andthe other PCR primer possesses a 5′-phosphate group. The PCR generatedinsert molecule is generated and is first ligated to one vector arm, forexample, a LVA to create a LVA-I-5′-phosphate molecule. The 5′-phosphateend of this molecule is unable to ligate to the LVA or RVA because thevector arm sites to which the TOPO is bound contain a 3′-phosphate. Thismolecule is then dephosphorylated to create to LVA-I-5′-OH. TheLVA-I-5′-OH molecule is then ligated to the other vector arm (RVA) toform LVA-I-RVA (FIG. 2). Once the ligated insert/vector moleculedescribed above has been constructed, the two vector arms can benon-covalently or covalently joined to one another, at the ends distalto the covalently attached topoisomerase polypeptide (i.e., their freeends), by a number of methods such that a circular molecule is formed.Such methods include, for example, ligase enzyme mediated ligation,complementary sequence annealing, topoisomerase mediated ligation, invitro or in vivo site-specific recombination, or in vivo homologousrecombination.

Example 2

Directional Molecular Cloning Using Topoisomerase and a Ligase Enzyme

A nucleic acid insert is generated using, for example, a pair of PCRprimers wherein one primer (P1) has a hydroxyl group at its 5′-end(OH-P1) and the other primer (P2) has a phosphate group at its 5′-end(P2-P) (see FIG. 3). The insert molecule is generated by PCR. A linearvector nucleic acid is prepared such that it has TOPO bound at one end(univalent TOPO-bound nucleic acid molecule); the other end of thelinear vector nucleic acid comprises a substrate for ligation (a 3′-OH)to be mediated by a ligase enzyme. In a single incubation, the PCRinsert can be ligated to the TOPO-end of the linear vector nucleic acidvia TOPO-mediated ligation and to the other end of the linear vectornucleic acid via a ligase enzyme-mediated reaction. The product of theligation is transformed into an appropriate host cell. A cloning eventmediated by both topoisomerase and DNA ligase is unidirectional. Thehydroxyl or phosphate group at the 5′-end of the PCR primers determinesthe directionality of the insert.

A second approach involving a topoisomerase- and ligase-mediatedligation comprises generation of an insert by for example, PCR. WherePCR is used to generate an insert, a pair of PCR primers where one has ahydroxyl group at its 5′-end (HO-P1) and the other has a phosphate groupat its 5′-end (P2-P) (see FIG. 4). A vector, such as two vector nucleicacid arms, can be prepared such that one vector arm has a TOPO bound atone end (univalent TOPO-bound DNA molecule) and the other vector arm hasa substrate for ligation at one end. In a single incubation, the PCRinsert is ligated to the one vector arm with a TOPO end viaTOPO-mediated ligation and to the other vector arm with theligation-ready end via ligase enzyme-mediated reaction. The product ofthe ligation is transformed into an appropriate host cell. The cloningevent mediated by both topoisomerase and DNA ligase is unidirectional.The hydroxyl or phosphate group at the 5′-end of the PCR primersdetermines the directionality. The other ends of the two vector arms arethen joined by any of the methods described above. Using this cloningmethod the ligation products comprised of RVA-I-RVA or LVA-I-LVA shouldnot be formed, but in the event that some do occur, such ligationproducts are incapable of subsequent replication and propagation.

Example 3

Molecular Cloning Using Topoisomerase and cos Ends

A method of molecular cloning using topoisomerase and cos ends cancomprise a vector, where such a vector may consist of two vector arms,with each arm consisting of one TOPO-end and one cos end. cos refers tothe cohesive ends present at the termini of bacteriophage lambda. Aninsert, such as a PCR insert, can be generated using primers comprising5′-OH termini. The PCR insert can be ligated to a TOPO-end of the twovector arms by DNA topoisomerase (see FIG. 5). Ligation events thatresult in LVA-I-LVA or RVA-I-RVA cannot subsequently be propagated. Theproduct of the ligation can be transformed into a suitable host. Thedistal ends of the vector arms contain terminal cos sites that arereadily annealed to one another in E. coli host cells by virtue of theirexplicit sequence. cos sites do not anneal in vitro at room temperature.

This method of cloning can be directional or non-directional. In thecase of non-directional cloning, an insert comprises a 5′-hydroxyl endsand can be ligated to, for example, two vector arms in a singlereaction. For directional cloning, an insert can be generated by, forexample, PCR wherein one PCR primer has a 5′-hydroxyl group and theother PCR primer has a 5′-phosphate group. Thus, the resulting PCRinsert will contain one 5′-hydroxyl end and one 5′-phosphate end. Theinsert is to be ligated sequentially, first to a left vector armcontaining a TOPO bound end followed by dephosphorylation of the5′-phosphate of the insert and then ligation to the right vector armcontaining a TOPO bound end (FIG. 6).

The ligation product of the insert to the vector is a linear molecule invitro with two cos sequences at its end. It is transformed into a host,such as E. coli more efficiently than a circular molecule.

Example 4

Molecular Cloning Using Topoisomerase and LIC Ends

A method of molecular cloning using topoisomerase and LIC ends cancomprise a vector, such as two vector arms, each consisting of oneTOPO-end and one LIC end. An insert, such as a PCR insert, can begenerated using primers comprising two 5′-OH termini. The PCR insert canbe ligated to a TOPO-end of the two vector arms by DNA topoisomerase(see FIG. 7). Ligation events that result in LVA-I-LVA or RVA-I-RVAcannot subsequently be propagated. The distal ends of the vector armscontain terminal LIC sites that are readily annealed to a plasmidcomprising LIC compatible ends.

This method of cloning can be directional or non-directional. In thecase of non-directional cloning, an insert comprising 5′-hydroxyl endsand can be ligated to, for example, two vector arms in a singlereaction. For directional cloning, an insert can be generated by, forexample, PCR wherein one PCR primer has a 5′-hydroxyl group and theother PCR primer has a 5′-phosphate group. Thus, the resulting PCRinsert will contain one 5′-hydroxyl end and one 5′-phosphate end. Theinsert is to be ligated sequentially, first to the left vector armcontaining a TOPO bound end and followed by dephosphorylation of the5′-phosphate of the insert and then ligation to the right vector armcontaining a TOPO bound end (FIG. 8).

Example 5

Molecular Cloning into Lambda Vector

The vector can comprise lambda DNA vector arms (termed left lambda arm(LLA)) and right lambda arm (RLA)). An insert, such as a PCR generatedinsert, can be ligated to the lambda vector arms in a directional manneror non-directional manner. In the case of non-directional cloning, a PCRinsert can be generated using 5′-hydroxyl PCR primers. The insert can beligated to two lambda vector arms in a single reaction. Ligation eventsresulting in LLA-I-LLA or RLA-I-RLA cannot subsequently be propagated.For directional cloning, one PCR primer has a 5′-hydroxyl end and theother PCR primer has a 5′-phosphate end. Thus, the PCR insert iscomprised of one 5′-hydroxyl end and one 5′-phosphate end. The insertcan be ligated sequentially to the two lambda vector arms with adephosphorylation step in between as depicted in FIG. 9. The ligatedlambda construct can be packaged in vitro and transfected into hostcells such as XLI-Blue E. coli. A circular plasmid DNA containing theinsert of interest can be rescued from the lambda vector using, forexample, ZAP technology (Stratagene).

Example 6

Molecular Cloning Into a Linear Plasmid DNA Molecule

A vector can comprise vector arms of a linear plasmid such as N15. Aninsert, such as a PCR generated insert, can be ligated to the plasmidvector arms in a directional manner or non-directional manner. In thecase of non-directional cloning, a PCR insert can be generated using5′-hydroxyl PCR primers. The insert can be ligated to two plasmid vectorarms in a single reaction (FIG. 10). Ligation events resulting inLVA-I-LVA or RVA-I-RVA cannot subsequently be propagated. Fordirectional cloning, one PCR primer has a 5′-hydroxyl end and the otherPCR primer has a 5′-phosphate end. Thus the PCR insert is comprised ofone 5′-hydroxyl end and one 5′-phosphate end. The insert can be ligatedsequentially to the two plasmid vector arms with a dephosphorylationstep in between as depicted in the FIG. 11. The linear DNA can betransformed directly into E. Coli. Alternatively, the ligated plasmidconstruct can be packaged in vitro and transfected into host cells suchas XLI-Blue E. coli. A DNA containing the insert of interest can berescued from the vector using, for example, ZAP technology (Stratagene).

A vector can also comprise a linear plasmid vector consisting of acovalently bound topoisomerase polypeptide at one end and a ligationsubstrate site at the other end (see FIG. 4). Incubation of the vectorwith an insert molecule comprising 5′-OH group on one end and a5′-phosphate group on the other end, under conditions sufficient fortopoisomerase-mediated ligation and ligase enzyme-mediated ligationresults in a ligated circular plasmid comprising the insert molecule.The plasmid can be transformed into a host cell.

Example 7

Molecular Cloning Using Topoisomerase and Site-Specific Recombination

A vector can comprise vector arms that comprise one TOPO-end and oneloxP end. The loxP site can be recombined with a second loxP site in thepresence of a Cre site-specific recombination protein. An insert, suchas a PCR generated insert, can be ligated to the TOPO-end of the twovector arms. Such cloning can be directional or non-directional. In thecase of non-directional cloning, an insert, such as a PCR insert can begenerated from PCR primers each comprising 5′-hydroxyl ends. An insertcomprising two 5′-OH ends can be ligated to two vector arms in a singlereaction (FIG. 12). For directional cloning, an insert can be generatedby, for example, PCR wherein one PCR primer comprises a 5′-hydroxyl endand the other PCR primer comprises 5′-phosphate end resulting in aninsert that comprises one 5′-hydroxyl end and one 5′-phosphate end. Theinsert can be ligated sequentially to two vector arms with adephosphorylation step in between as depicted in FIG. 13. The ligationproduct comprises a loxP site at each end of a linear molecule. Thelinear molecule can be recombined into a circular recombinant plasmid invitro, for example using purified Cre recombinase or in vivo by, forexample transformation into an E. coli host expressing Cre recombinaseand a plasmid that has loxP sites.

Example 8

Molecular Cloning Using Topoisomerase and Homologous Recombination inVivo

In vivo homologous recombination can be exploited to transfer a ligatedinsert/vector of interest into a circular plasmid vector. Homologoussequences flank a ligated insert/vector of interest and aresubstantially identical to sequences of a plasmid cloning vector. Aligated insert/vector of interest is recombined into a plasmid cloningvector of choice via homologous recombination between the homologoussequences flanking the ligated insert/vector and in the plasmid cloningvector. An insert can be generated with homologous sequences attached toeach end by, for example, synthesizing PCR primers with homologousvector sequences, of for example, 30, 75, 100, 150, 200, 250, 500, or1000 base pairs and using the PCR primers to generate a ligatedinsert/vector with homologous vector sequences flanking the ligatedinsert/vector of interest. A ligated insert/vector molecule withhomologous sequences at the ends can also be generated by preparingtopoisomerase-bound homologous sequence elements and employing a TOPOcloning scheme as outlined in FIGS. 14 and 15 for generating an insertwith homologous sequence elements on each end. A PCR amplified insertcontaining TOPO ligated arms can be transformed into host cellscontaining a cloning vector wherein homologous recombination can occur.For efficient in vivo homologous recombination, a recA+ host strain canbe used. To protect a linear insert from degradation by endogenousexonuclease activities, the ends of the insert can be modified to eitherinhibit or prohibit exonuclease digestion events.

To achieve site-specific in vivo recombination, lambda attachment sitescan be employed in place of the homologous sequences described above. Inthis scenario, lambda attachment sites flank a ligated insert/vector ofinterest, which is generated according to the PCR and TOPO cloningschemes described above. The ligated insert/vector with the flankinglambda attachment sites is transformed into host cells containing acloning vector with lambda attachment sites. Inside the host cell, theligated insert/vector then can be site-specifically recombined into aplasmid cloning vector between the lambda attachment sites flanking theligated insert/vector and those sites in the plasmid cloning vector.

OTHER EMBODIMENTS

Other embodiments are within the following claims.

1. A method of covalently joining a nucleic acid insert molecule tofirst and second nucleic acid flanking molecules to form a ligatedmolecule, the method comprising (A) incubating: said insert molecule,wherein one end of said insert molecule comprises a 5′-hydroxyl groupand the other end comprises a 5′-phosphate group, and a said firstflanking molecule, wherein one end only of said first flanking moleculecomprises a covalently bound topoisomerase polypeptide, under conditionswhich permit their covalent joining to form a ligated nucleic acidcomprising a said insert molecule positioned adjacent to a said firstflanking molecule, (B) incubating a said ligated nucleic acid of step(A) with phosphatase under conditions which permit removal of a5′-phosphate group from said ligated nucleic acid; and (C) incubating aproduct of step (B) with a said second flanking molecule, wherein oneend only of said second flanking molecule comprises a covalently boundtopoisomerase polypeptide, under conditions which permit covalentjoining to form a ligated molecule comprising a said insert moleculepositioned between a said first and a said second flanking molecule. 2.The method of claim 1 wherein a said first and a said second nucleicacid flanking molecules comprise a left and a right vector arm,respectively, such that a said insert molecule is flanked by a said leftvector arm and a said right vector arm.
 3. The method of claim 2,wherein said left and right vector arms each comprise a free end that isnot joined to an insert molecule, said method further comprising thestep of: joining the free ends of said vector arms to each other by amethod selected from the group consisting of nucleic acid ligasemediated ligation, complementary sequence annealing, topoisomerasemediated ligation, in vitro site-specific recombination, in vivosite-specific recombination, and in vivo homologous recombination.
 4. Amethod of covalently joining a nucleic acid insert molecule to first andsecond nucleic acid flanking molecules to form a ligated molecule, themethod comprising incubating: said insert molecule and said flankingmolecules, wherein one end of said insert molecule comprises a5′-hydroxyl group and the other end comprises a 5′-phosphate group,wherein one end only of said first flanking molecule comprises acovalently bound topoisomerase polypeptide and wherein one end of saidsecond flanking molecule comprises a ligase substrate site, underconditions which permit their covalent joining to form a ligatedmolecule comprising a said insert molecule positioned between a saidfirst and a said second flanking molecule.
 5. The method of claim 4wherein a said first and a said second nucleic acid flanking moleculescomprise a left and a right vector arm, respectively, such that a saidinsert molecule is flanked by a said left vector arm and a said rightvector arm.
 6. The method of claim 5, wherein said left and right vectorarms each comprise a free end that is not joined to an insert molecule,said method further comprising the step of: joining the free ends ofsaid vector arms to each other by a method selected from the groupconsisting of nucleic acid ligase mediated ligation, complementarysequence annealing, topoisomerase mediated ligation, in vitrosite-specific recombination, in vivo site-specific recombination, and invivo homologous recombination.